Mechanistic understanding of living things requires our understanding of how proteins and DNA interact together to generate functional chromosomes. This understanding is central to preserving human health, dealing with genetic disorders, and fighting pathogenic organisms. The proposed projects are focused on single-molecule analyses which permit direct visualization of biomolecule interactions, and can be used to analyze protein-DNA interactions in detail. The aims of the proposal include careful study of an exchange mechanism for removal of proteins from DNA that suggests a major revision of conventional descriptions of protein turnover on DNA, and which will affect a wide range of studies of gene regulatory and chromosome-structural proteins. A second aim is focused on direct study of mechanisms of large Structural Maintenance of chromosomes protein complexes which mediate the folding of chromosomes in eukaryote cells. Finally, a third aim is focused on mechanisms underlying a family of cut and paste DNA recombination systems responsible in part for generating bacteria genetic diversity. The highly mechanistic analyses of DNA-processing machinery that are proposed will give us a stronger understanding of how cells interpret, fold and change their genomes, leading to a better understanding of pathologies where those functions are impaired, and better understanding of how to target those functions in pathogenic organisms.